title: Diversity in functional organization of class I and class II biotin protein ligase.
abstract: The cell envelope of Mycobacterium tuberculosis (M. tuberculosis) is composed of a variety of lipids including mycolic acids, sulpholipids, lipoarabinomannans, etc., which impart rigidity crucial for its survival and pathogenesis. Acyl CoA carboxylase (ACC) provides malonyl-CoA and methylmalonyl-CoA, committed precursors for fatty acid and essential for mycolic acid synthesis respectively. Biotin Protein Ligase (BPL/BirA) activates apo-biotin carboxyl carrier protein (BCCP) by biotinylating it to an active holo-BCCP. A minimal peptide (Schatz), an efficient substrate for Escherichia coli BirA, failed to serve as substrate for M. tuberculosis Biotin Protein Ligase (MtBPL). MtBPL specifically biotinylates homologous BCCP domain, MtBCCP(87), but not EcBCCP(87). This is a unique feature of MtBPL as EcBirA lacks such a stringent substrate specificity. This feature is also reflected in the lack of self/promiscuous biotinylation by MtBPL. The N-terminus/HTH domain of EcBirA has the self-biotinable lysine residue that is inhibited in the presence of Schatz peptide, a peptide designed to act as a universal acceptor for EcBirA. This suggests that when biotin is limiting, EcBirA preferentially catalyzes, biotinylation of BCCP over self-biotinylation. R118G mutant of EcBirA showed enhanced self and promiscuous biotinylation but its homologue, R69A MtBPL did not exhibit these properties. The catalytic domain of MtBPL was characterized further by limited proteolysis. Holo-MtBPL is protected from proteolysis by biotinyl-5' AMP, an intermediate of MtBPL catalyzed reaction. In contrast, apo-MtBPL is completely digested by trypsin within 20 min of co-incubation. Substrate selectivity and inability to promote self biotinylation are exquisite features of MtBPL and are a consequence of the unique molecular mechanism of an enzyme adapted for the high turnover of fatty acid biosynthesis.
authors.affiliation: Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India.
authors.forename: Sudha
authors.initials: S
authors.lastname: Purushothaman
authors.forename: Karthikeyan
authors.initials: K
authors.lastname: Annamalai
authors.forename: Anil K
authors.initials: AK
authors.lastname: Tyagi
authors.forename: Avadhesha
authors.initials: A
authors.lastname: Surolia
mesh_terms: D000103:Acetyl-CoA Carboxylase / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D001710:Biotin / Q000378:metabolism; D019921:Biotinylation; D019731:Carbon-Nitrogen Ligases / Q000737:chemistry / Q000378:metabolism*; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D054889:Fatty Acid Synthase, Type II / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D010455:Peptides / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D010455:Peptides; D012097:Repressor Proteins; D001710:Biotin; D054889:Fatty Acid Synthase, Type II; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli; D000103:Acetyl-CoA Carboxylase; C079633:biotin carboxyl carrier protein